Analyzing a Complex Sample by MS/MS Using Isotopically-Labeled Standards

ABSTRACT

A method and corresponding apparatus are disclosed for analysis of a peptide-containing sample. The sample is prepared by adding isotopically-labeled peptides corresponding to endogenous peptides of interest, and the prepared sample is analyzed by liquid chromatography-mass spectrometry (LCMS). Detection in a high-resolution, accurate mass (HRAM) MS1 spectrum of a precursor ion matching an isotopically-labeled peptide triggers acquisition of an MS/MS spectrum (preferably acquired in an ion trap or other fast mass analyzer) to determine if a product ion is present matching a characteristic product ion (e.g., the y1 ion) of the isotopically-labeled peptide. If the characteristic product ion is present, then a HRAM MS/MS spectrum is acquired for detection and quantitation of the corresponding endogenous peptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation under 35 U.S.C. § 120 andclaims the priority benefit of co-pending U.S. patent application Ser.No. 15/164,113, filed May 25, 2016. The disclosure of the foregoingapplication is incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to mass spectrometry, and moreparticularly to targeted methods of analyzing a complex sample by MS/MSanalysis using isotopically labeled standards.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted concurrently withthe specification as a text file via EFS-Web, in compliance with theAmerican Standard Code for Information Interchange (ASCII), with a filename of TP20091US2-NAT_ST25.txt, a creation date of Apr. 6, 2018, and asize of 2.73 KB. The sequence listing filed via EFS-Web is part of thespecification and is hereby incorporated in its entirety by referenceherein.

BACKGROUND OF THE INVENTION

Protein analysis by mass spectrometry is largely done via a bottom-up,data-dependent method. With this approach, proteins are first digestedby enzymes into peptides. These peptides are then separated either byone or more dimensions of liquid chromatography. Following separation,the eluting peptides are ionized and analyzed with the mass spectrometerusing an MS1 “survey scan”. From this initial mass spectral analysis,precursors are selected for subsequent analysis using a set of criteria(e.g., precursor charge state). These selected precursors are furtherinterrogated by MS/MS analysis (e.g., via isolation, fragmentation, andm/z analysis).

This approach is quite effective at covering a very wide range ofpeptides in a very short amount of time. However, due to the limiteddynamic range of the MS1 analysis, and the stochastic nature ofdata-dependent precursor sampling, the depth and reproducibility of thisapproach is often poor. For example, it is rare that this approachprovides complete coverage of biological functional groups or pathways(e.g., it is unlikely a data-dependent analysis would cover all thehuman kinases). These workflow limitations often result in poor overlapbetween replicate experiments.

Targeted proteomics approaches, such as selected reaction monitoring(SRM), multiple reaction monitoring (MRM), and parallel reactionmonitoring (PRM), are the traditional alternatives to the data-dependentapproach. In lieu of selecting precursors from an MS1 survey spectrum,the instrument dwells upon select m/z regions that are informed by alist that is populated by the user before the analysis. That is, theinstrument continuously collects MS/MS spectra, independent of whetherthere is any detectable signal in an MS1 spectrum. By specificallydwelling on prespecified precursors, these methods are capable of muchhigher sensitivity and reproducibility than the data-dependent workflow.

However, one of the main compromises with these targeted analyses isbreadth. Dwelling upon low abundance precursors comes at the cost ofinterrogating higher abundance species. Also, without any pre-screeningof the eluting peptides (i.e., MS1 survey spectra), it is expected thatsome portion of the targeted MS/MS analyses will occur when there is noprecursor present (i.e., MS/MS spectra will be collected while theprecursor isn't eluting). These concerns can be somewhat mitigated byemploying complex retention time scheduling—that is, along with the listof precursor targets the user provides a list of retention times.However, scheduling targeted scans in this manner requires preciseknowledge of peptide retention times. These times are specific to thechromatographic setup, and the utility of these times are heavilycontingent upon the reproducibility of the chromatographic separation.

As an alternative to these traditional approaches, two labs havepublished workflows that attempt to realize the depth andreproducibility of the targeted workflow while still leveraging the easeof use of the data-dependent approach (see Gallien et al., “Large-ScaleTargeted Proteomics Using Internal Standard Triggered-Parallel ReactionMonitoring (IS-PRM)”, Mol. Cell. Proteomics, Vol. 14, No. 6, pp. 1630-44(2015); Yan et al., “Index-ion Triggered MS2 Ion Quantification: A NovelProteomics Approach for Reproducible Detection and Quantification ofTargeted Proteins in Complex Mixtures”, Mol. Cell. Proteomics, Vol. 10,No. 3, M110.005611 (2011)). These workflows begin with the addition ofheavy peptide standards to the analytical sample. These heavy standardshave sequences that are analogous to endogenous peptides of interest. Byincorporating specific heavy isotopes, these standards differ in massfrom the endogenous form of the peptide; however, their retention timesmatch exactly. In the published workflows, the mass spectrometer methodincludes low-quality targeted scans on the spiked-in standards.Following acquisition of the targeted MS/MS transitions/spectra, thedata is analyzed, and if certain conditions are met the instrumenttriggers a high-quality MS/MS analysis on the expected location of theendogenous form of the peptide.

While these workflows have been partially successful at addressing thelimitations and disadvantages of the established data-dependent andtargeted methods, they tend to have a steep tradeoff between sensitivityand selectivity, with one of the published workflows offering goodsensitivity but limited selectivity, and the other exhibiting excellentselectivity with modest sensitivity. Furthermore, both workflows rely onretention time scheduling to achieve acceptable duty cycle andselectivity, which increases method setup complexity, places stress onsample availability, and can compromise robustness, particularly whererun-to-run variation of chromatographic separation exits.

SUMMARY

Roughly described, embodiments of the present invention adopt anapproach in which detection of a characteristic product ion derived fromdissociation of an isotopically-labeled peptide ion triggers MS/MSanalysis of the corresponding endogenous peptide ion. More specifically,a method is provided for analysis of a peptide-containing sample whereinthe sample is spiked with a plurality of isotopically-labeled peptides,at least some of which are labeled forms of endogenous peptides ofinterest. According to one example, the labeled peptides aremetabolically labeled with heavy forms of the C-terminal lysine orarginine. The sample is subjected to chromatographic separation and theeluting components are analyzed by mass spectrometry according to aprescribed sequence of steps. An MS1 survey scan is performed,preferably in a high-resolution mass analyzer such as an orbitalelectrostatic trap, and the resultant MS1 spectrum is processed toidentify precursor ions in the spectrum that have mass-to-charge ratios(m/z) that match an m/z that corresponds to one of theisotopically-labeled peptides. If a match is identified, then a firstMS/MS scan is performed to acquire a spectrum of product ions generatedby dissociation of the matching precursor ion appearing in the MS1spectrum. In order to provide high sensitivity and throughput, the firstMS/MS scan may be performed in a fast and sensitive mass analyzer, suchas a quadrupole ion trap. The speed of the first MS/MS scan may beincreased by limiting the scan range to a narrow m/z window around thevalue of a characteristic product ion of the isotopically labeledpeptide, for example the y₁ product ion. If the MS/MS spectrum exhibitsa peak of sufficient intensity (indicating with a high degree ofconfidence the presence of the characteristic product ion of theisotopically-labeled peptide), then one or a series of MS/MS scans areperformed to quantify the endogenous peptide ion corresponding to thelabeled peptide ion. The MS/MS scan of the endogenous peptide may beconducted in the high-resolution (e.g., orbital electrostatic trap) massanalyzer.

The foregoing method presents several advantages relative to the priorart approaches described above, including achieving very goodselectivity while retaining high sensitivity, and avoiding the need forretention-time scheduling and/or other techniques that complicate methoddevelopment and render the method vulnerable to problems arising fromrun-to-run variations.

BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings:

FIG. 1 is a symbolic diagram depicting a liquid chromatography/massspectrometry (LC/MS) system configurable to perform analysis ofpeptide-containing samples in accordance with embodiments of the presentinvention; and

FIG. 2 is a flowchart depicting steps of a method for analysis ofpeptide-containing samples by mass spectrometry;

FIG. 3 is a table containing an inclusion list of m/z values of labeledpeptide ions, used for matching of precursor ions appearing in an MSspectrum; and

FIG. 4 depicts ion chromatograms corresponding to anisotopically-labeled peptide and its endogenous equivalent acquiredusing the FIG. 2 method.

DETAILED DESCRIPTION OF EMBODIMENTS

FIG. 1 depicts an LC/MS system 100 that may be employed to implementembodiments of the present invention. The description of LC/MS system100 is intended to be illustrative of an exemplary implementation, andshould not be construed as limiting the invention to any particularinstrument architecture. LC/MS system 100 includes a liquidchromatography (LC) system 110 coupled to a mass spectrometer 120. LCsystem 110 may be conventionally provided with pumps for loading thesample onto a chromatographic column and for directing one or moresolvents onto the column according to an elution gradient to causecomponents of the sample to be chromatographically separated, such thatthey are eluted from the column at different retention times. The eluatefrom LC system 110 is passed to an ionization source 130 of massspectrometer 120, which produced gas-phase ions from molecules,including biological molecules such as peptides, contained in thesample. The ionization source may be of any suitable type known in theart, such as an electrospray ionization (ESI) source. The ions producedin source 130 are delivered via ion optics 140 (which will typicallyinclude some combination of ion lenses and ion guides designed toconfine and focus the ions to an ion path axis) to a quadrupole massfilter (QMF) 150, which is operable, by adjustment of the amplitudes ofoscillatory and resolving direct current (DC) voltage applied to itselectrodes, to selectively transmit only ions within a desired range ofmass-to-charge (m/z) values. The ions thus transmitted (which maycomprise a single ion species or multiple ion species) are passed into agas-filled collision cell 160, where the ions may undergo collisionswith atoms or molecules of gas resulting in fragmentation into productions via collisionally activated dissociation (CAD). Collision cell 160may also function to route ions transmitted thereto and/or formedtherein by CAD to a selected one of the first mass analyzer 170 orsecond mass analyzer 180. In one example of mass spectrometer 120, firstmass analyzer 170 is an orbital electrostatic trap mass analyzer (e.g.,a mass analyzer of the type sold by Thermo Fisher Scientific Inc. underthe trademark “Orbitrap”), and second mass analyzer 180 is a quadrupoleion trap (e.g., a two-dimensional orthogonal-ejection ion trap). Eachmass analyzer functions to separate ions according to their m/z's suchthat a mass spectrum may be acquired. The various components of massspectrometer 150 communicate with and are controlled by controller 190,which also serves to record and process mass spectral data generated bythe mass analyzers. Although controller 190 is depicted symbolically asa single unit, its functions will typically be distributed across anumber of physically separate devices, including but not limited tospecialized processors, storage, memory, general-purpose computers andapplication-specific circuitry. Controller 190 is adapted with a userinterface, enabling the user to specify experimental parameters or otherinformation, and to view and manipulate results. Controller 190 isfurther configured with executable instructions, typically in the formof software code, for performing certain of the steps of the methodsdescribed below.

The steps of a method for analyzing peptide-containing samples inaccordance with embodiments of the present invention may be understoodwith reference to the FIG. 2 flowchart. The method includes an initialstep 210 of preparing a “spiked” sample that incorporates one or moreisotopically-labeled (“heavy”) peptides corresponding to targetedendogenous peptides. Typically, the sample will take the form of abiological fluid (e.g., blood plasma, saliva, urine, cerebrospinalfluid) or tissue extract. The sample may be subjected to one or morepreparation steps known in the art, either prior to or followingenzymatic digestion, to remove unwanted components, concentrate orpurify components of interest, and to facilitate analysis by LC/MS.According to the approach commonly known as “bottom-up” analysis,proteins contained in the sample may be digested to form peptides by theaddition of a proteolytic enzyme, such as trypsin.

Following or concurrently with other sample preparation steps, a set ofisotopically-labeled peptides are added in known quantities to thesample. At least some of the added isotopically-labeled peptides arelabeled versions of the endogenous peptides of interest (i.e., those tobe detected and quantified in the sample). In one example, if theendogenous peptides present in the sample are tryptic peptides (notingthat trypsin cleaves proteins next to arginine (R) or lysine (K)), thenthe isotopically-labeled peptides may be metabolically labeled withheavy forms of C-terminal arginine or lysine. For example, if it isdesired to measure the amount of peptide IGDYAGIK (SEQ ID NO:1) in asample, then the set of isotopically-labeled peptides added to thesample may include IGDYAGIK* (SEQ ID NO:2), where K* represents theheavy (fully ¹³C and ¹⁵N substituted) form of the terminal lysine aminoacid, such that the isotopically-labeled version of the peptide has amass 8 Dalton (Da) greater than the endogenous (light) peptide. Inanother example, the endogenous peptides of interest include SSAAPPPPPR(SEQ ID NO:3), and the isotopically-labeled peptides include SSAAPPPPPR*(SEQ ID NO:4), where R* is the fully ¹³C and ¹⁵N substituted form of theterminal arginine amino acid, yielding an isotopically-labeled versionof the peptide having a mass 10 Da greater than the endogenous (light)peptide. This labeling scheme enables confirmation of the presence of alabeled peptide ion by detection of a characteristic product ionincorporating the labeled amino acid, such as the y₁ ion, as isdiscussed below.

It should be recognized that the present invention is not limited to theexample presented above, in which the added standard consist of peptideshaving metabolically labeled terminal arginine or lysine. In alternativeimplementations of the invention, the labeled peptides may bemetabolically labeled with heavy versions of other amino acids, or thelabeled peptides may be labeled via a chemical tagging approach.Alternatively the sample of endogenous peptides may be labeled, whilethe spiked-in standard contains isotopes of natural abundances.

In another variant of the sample preparation method,isotopically-labeled intact proteins, corresponding to endogenous intactproteins of interest, are added to he sample prior to enzymaticdigestion. The endogenous and isotopically-labeled proteins in thesample are then digested concurrently (e.g., by the addition of trypsinor other proteolytic enzyme) to yield endogenous andisotopically-labeled peptides, which serve as surrogates to thecorresponding proteins, and may be analyzed in accordance with the massspectrometry-based technique described below.

It is further noted that the labeling scheme utilized to produce theisotopically-labeled peptides should be selected in view of variousperformance factors relating to the mass spectrometry analysis methoddescribed below. More specifically, the method relies on the detectionof characteristic product ions of the isotopically-labeled peptide toinitiate quantitative measurement of the corresponding endogenouspeptide. To improve specificity and avoid spurious triggering ofquantitation scans, it is helpful to select an isotopic labeling schemethat will yield characteristic product ions from the labeled speciesthat have m/z's sufficiently different from potentially interferingproduct ions generated by fragmentation of isobaric precursor ionspecies, e.g., precursor ions produced by ionization of peptides andother compounds that are not of interest.

The spiked sample, incorporating the isotopically-labeled peptides, isthen chromatographically separated in step 220 into constituentcomponents, e.g., by passing the spiked sample into a chromatographiccolumn and gradually eluting components according to theirhydrophobicities via a suitable elution gradient. The endogenous andlabeled versions of a peptide have substantially identicalhydrophobicities and elute from the column simultaneously. As thecomponents elute from the column, they are passed to ionization source130, which ionizes the sample components to form ions, including ions ofthe labeled peptides and corresponding endogenous peptides contained inthe sample. The ions thus generated are then analyzed by massspectrometry in accordance with the steps depicted in FIG. 2 anddiscussed below.

In step 230, an MS1 scan (also referred to as a “survey scan”) isperformed to acquire a mass spectrum of the precursor (intact) ionsformed by ionization of the sample. In one embodiment, the MS1 scan isacquired in a mass analyzer, such as an orbital electrostatic trap massanalyzer, capable of acquiring high resolution/accurate mass (HRAM)spectral data, for example at a resolving power (at 200 m/z) exceeding50,000 and at a mass accuracy better than 10 parts per million (ppm).The mass spectrum will typically include one or more peaks or features(referred to herein as precursor ion peaks) indicating the presence ofions at specific values of m/z. Each precursor peak will have a heightor intensity that indicates the abundance of ions at the peak m/z. Incertain implementations, the mass spectrum may be filtered to removepeaks that cannot be reliably distinguished from noise, e.g., bydiscarding peaks from the spectrum that do not meet a minimum intensityor signal-to-noise threshold.

The mass spectrum (or a filtered version thereof) is processed tocompare m/z values of peaks appearing in the mass spectrum to a storedlist of m/z values corresponding to ions of the labeled peptides todetermine whether any of the peaks match (i.e., fall within a masswindow surrounding) values on the stored list, step 240. An example of atable containing a stored list of m/z values, and their correspondingpeptide sequences and charge states, is depicted in FIG. 3. The masswindow used for matching peaks to values in the stored list may be set,either manually or automatically, in view of the mass accuracy of themass analyzer as well as a desired degree of selectivity (i.e., the masswindow may be set to a particularly narrow range to minimize spuriousidentifications arising from interfering ions having m/z values close tothose of the labeled peptide ions). In certain embodiments, the storedlist may be dynamically adjusted to account for expected elution times,i.e., at a particular moment in chromatographic time, the list mayinclude the m/z values of only those labeled peptides that would beexpected to be within their chromatographic elution window. In order tofurther avoid spurious identification, additional criteria may beapplied to assess whether a labeled peptide ion is genuinely present;such criteria may include, for example, the presence/intensity of peaksat other expected charge states of the labeled peptide ion.

If no precursor ion peaks are found in the mass spectrum that matchvalues on the stored list and satisfy other (optional) criteria,indicating that no labeled peptides are eluting at that timepoint, themethod reverts to step 230 for acquisition of another MS1 spectrum.

If it is determined in step 240 that a precursor ion peak in the MS1spectrum matches a value on the stored list, then the method proceeds tostep 250, whereby MS/MS analysis is conducted to identify the presenceof a characteristic product ion or product ions generated byfragmentation of the labeled peptide ion. According to the examplepresented above, wherein IGDYAGIK (SEQ ID NO:1) is a peptide ofinterest, a precursor ion peak of sufficient intensity may be identifiedin the MS spectrum at an m/z of 422.7363, matching (i.e., within thespecified mass window of) the value on the stored list (FIG. 3)corresponding to the labeled peptide IGDYAGIK* (SEQ ID NO:2). In step240, the precursor ions at the matching m/z value are mass isolated(i.e., separated from ions of other masses) and subjected to controlleddissociation to yield product ions. In the FIG. 1 example, isolation ofthe precursor ions may be effected by setting the amplitudes of the RFand resolving DC voltages of QMF 150 to values that provide selectivetransmission of ions having masses lying within a narrow window of thetheoretical mass of the labeled peptide ions. In an illustrativeimplementation, QMF 150 is tuned to selectively transmit precursor ionsin a 0.7 m/z wide window centered on the m/z value of the isotopicallylabeled peptide precursor ion; in the foregoing example, thetransmission window may be set to 422.7363±0.35 m/z. Alternatively,isolation may be performed in a quadrupole ion trap, e.g., second massanalyzer 180, by application of a notched broadband waveform. Followingisolation, the precursor ions are dissociated (fragmented) to generateproduct ions. In one embodiment, dissociation of the precursor ions isachieved by the collisionally activated dissociation (CAD) technique,whereby the ions are collided at high energies with atoms or moleculesof a collision gas, such as nitrogen or argon. CAD may be effected byaccelerating a beam of precursor ions into gas-filled collision cell160. In another implementation, collisionally activated dissociation maybe performed by kinetically exciting precursor ions within a quadrupoleion trap via application of an excitation voltage to the trapelectrodes.

In alternative embodiments, dissociation techniques other than CAD maybe utilized for production of fragment ions. Such dissociationtechniques include electron transfer dissociation (ETD), pulsed-qdissociation (PQD) and photodissociation.

The product ions resulting from isolation and fragmentation of theprecursor ion are then mass analyzed to generate an MS/MS spectrum. Inan illustrative embodiment, the MS/MS spectrum is acquired by performingan analytical scan in an ion trap mass analyzer, e.g., second massanalyzer 180. Utilization of an ion trap mass analyzer may beparticularly advantageous due to its ability to acquire mass spectrarapidly and at high sensitivity. As is known in the art, an analyticalscan may be effected in an ion trap mass analyzer by the resonantejection method, wherein a resonant excitation voltage is applied to theion trap electrodes which the main RF trapping voltage is ramped, suchthat ions come into resonance with the resonant excitation field and areejected to a detector in order of their m/z's.

In order to minimize the duration of the analytical scan and therebyincrease the overall acquisition rate, the RF voltage ramp may belimited to a narrow range centered around the value corresponding to theejection voltage of the characteristic product ion. In the exampledescribed below, the characteristic product ion is the y₁ ion of theIGDYAGIK* (SEQ ID NO:2) precursor ion, having an m/z of 155.1, the scanrange width may be limited to 2 m/z, extending between 154 and 156 m/z.

The MS/MS spectrum from step 250 may then be processed in step 260 todetermine the presence/intensity of a peak corresponding to acharacteristic product ion of the isotopically-labeled precursor ion.According to one implementation, the characteristic product ion is they₁ ion. Per standard nomenclature in the mass spectrometry art, a y₁ ionis the C-terminal fragment ion containing a single amino acid producedby cleavage of a C—N bond in the peptide chain. It is noted thatcollisionally activated dissociation of peptide ions yields primarilyy-type and b-type product ions (complementary N-terminal fragment ionsproduced by C—N bond cleavages). In accordance with the labeling schemedescribed above, wherein the isotopically-labeled peptides aremetabolically labeled with heavy forms of C-terminal lysine or arginine,the y₁ ion will consist of protonated heavy forms of lysine (155.1 m/z)or arginine (185.1 m/z). For the IGDYAGIK* (SEQ ID NO:2) precursor ionpresented above as an example, the y₁ ion constitutes protonated heavylysine.

In a basic implementation, the determination in step 260 will involvedetermining whether a peak of sufficient intensity (i.e., above athreshold value) is present in the MS/MS spectrum within a specifiedwindow of the m/z value of the characteristic product ion (e.g., y₁) ofthe isotopically-labeled peptide; if no peak is present, or the peakintensity does not meet the threshold criteria, then it is determinedthat the characteristic product ion is not present. Otherimplementations may base the determination in step 260 on whether themultiplicative product of the intensities of the product ion peak in theMS/MS spectrum and the corresponding precursor ion peak in the MS1spectrum exceed a threshold, or whether the ratio between theintensities of the product ion peak and the corresponding precursor peakmeet a threshold value or are within a specified range of values. Stillother implementations may determine whether the multiplicative productor summed intensities of two or more product ion peaks in the MS/MSspectrum exceed a threshold. While the y₁ ion of the labeled peptide isthe characteristic product ion in the foregoing example, those skilledin the art will recognize that other fragments incorporating theisotopically labeled moiety (e.g., the labeled amino acid) may beemployed as the characteristic product ion instead of or in conjunctionwith the y₁ ion.

It is possible that greater specificity may be achieved if thedetermination in step 260 involves processing of multiple peaks withinthe MS/MS spectrum to ascertain whether plural characteristic productions are present, rather than limiting the determination to a singlecharacteristic product ion, such as the y₁ ion. However, such enhancedspecificity will come at a cost of more difficult and complex methodsetup (since multiple characteristic product ion m/z's will need to bedetermined and stored for each of the isotopically-labeled peptidesadded to the sample), as well as possibly requiring longer MS/MS scantimes. For this reason, it may be particularly favorable to limit thenumber of characteristic product ions to one ion (e.g., the y₁ ion) or asmall plurality (e.g., two or three) of product ions, such as the y₁ ionand one or two additional characteristic product ions).

If it is determined that there are no product ion peaks in the MS/MSspectrum that match the m/z value of the characteristic product ion,then the method returns to step 230 for acquisition of an MS1 spectrum.

If, however, the MS/MS spectrum exhibits a product ion peak ofsufficient intensity at the value of the characteristic product ion,then the method proceeds to step 270, whereby MS/MS analysis isconducted to measure the intensity of a product ion peak produced bydissociation of the endogenous peptide precursor ion corresponding tothe labeled peptide ion matched in the MS1 spectrum. In otherimplementations, step 270 may involve measuring the intensity of aplurality of characteristic product ions that are produced bydissociating the endogenous peptide. This measured intensity may beutilized to quantify the amount of the endogenous peptide ion, in themanner discussed below. According to one embodiment, the MS/MS spectrumfor step 270 is acquired in an HRAM mass analyzer, such as an orbitalelectrostatic trap mass analyzer (e.g., in first mass analyzer 170).Isolation of the endogenous peptide precursor ion, which has a mass thatis different from the corresponding labeled peptide ion by the number ofisotopic substitutions, may be accomplished by operation of thequadrupole mass filter to selectively transmit ions within a range ofm/z's occupied by the endogenous peptide precursor ion. Dissociation maybe effected by fragmenting the endogenous peptide precursor ions by CADin collision cell 160. The resultant product ions are mass analyzed togenerate an MS/MS spectrum.

According to one embodiment, step 270 includes isolating and fragmentingions matching the m/z's of both the endogenous peptide ion and itsisotopically labeled peptide counterpart, and collectively massanalyzing the product ions of each. In the above-described example, inwhich the endogenous peptide of interest is IGDYAGIK (SEQ ID NO:1),precursor ions corresponding to both the IGDYAGIK (SEQ ID NO:1)(endogenous) and IGDYAGIK*(SEQ ID NO:2) (isotopically-labeled) may beisolated and fragmented. Isolation and fragmentation of the two sets ofprecursor ions may be done sequentially, with subsequent combination ofthe product ions before mass analysis; alternatively, the two sets ofprecursor ions may be co-isolated (e.g., via use of a multi-notchisolation waveform, or by using an isolation width sufficiently broad toinclude the m/z's of both species) and the co-isolated precursor ionsmay be fragmented at the concurrently. Alternatively isolation,fragmentation, and analysis of the two sets of precursor ions may bedone sequentially, whereby we produce two spectra one containingfragment ions belonging to the endogenous peptide and the othercontaining fragment ions belonging to the isotopically-labeled species.In this manner, the MS/MS spectrum will include peaks corresponding toproduct ions of both the endogenous peptide ion and the correspondingisotopically-labeled peptide ion. Since the isotopically-labeledpeptides are added to the sample in known quantities, this enablesabsolute quantitation of the endogenous peptide using the ratios ofmeasured intensities of product ions in the MS/MS spectrum.

Following acquisition of the second MS/MS spectrum, comprising productions of the endogenous peptide ion and (optionally) of the “heavy”isotopically-labeled peptide ion, the method may return to step 230 foracquisition of an MS1 survey spectrum. Alternatively, a positivedetermination of the presence of the characteristic product ion in step260 may trigger the sequential execution of a number of MS/MSquantitation scans before returning to step 230. In either case, thedata in the MS/MS spectra acquired in step 270 may be used for relativeor absolute quantitation of the endogenous peptide of interest inaccordance with established methods, e.g., by integration of peaks inthe ion chromatograms corresponding to the product ion(s) of theendogenous peptide precursor ion and (optionally) of theisotopically-labeled peptide precursor ion. It is noted thatquantification may be done using the intensity of the counterpartproduct ion used for confirmation of the presence of theisotopically-labeled peptide in step (i.e., the y₁ ion of the endogenouspeptide precursor), or alternatively quantitation may be done using oneor more other product ions, or a combination of the product ion used forconfirmation and other product ions.

FIG. 4 presents examples of ion chromatograms obtained using a specificimplementation of the present method, in which a sample comprising 432isotopically-labeled peptides spiked into lung cancer cell digests wasanalyzed. The ion chromatograms depict peaks corresponding to theisotopically-labeled peptide SFLLALPAPLVTPEASAEAR* (SEQ ID NO:6) and itsendogenous counterpart SFLLALPAPLVTPEASAEAR (SEQ ID NO:5). The ionchromatogram in the lower left depicts, as a function of chromatographicretention time, the intensity of ions in the MS1 spectrum at the m/z ofthe SFLLALPAPLVTPEASAEAR* (SEQ ID NO: 6) precursor ion. It is observedthat a peak is present centered approximately at the retention time of54.8 minutes. The detection of precursor ions appearing in the spectrumat that m/z triggered MS/MS analysis of product ions, in accordance withthe method described above. The ion chromatogram at upper right depictsthe intensity of ions in the MS/MS spectrum at the m/z of the y₁ iongenerated by dissociation of the SFLLALPAPLVTPEASAEAR* (SEQ ID NO:6)precursor ion (185.1 m/z). This chromatogram also exhibits a peakcentered around 54.8 minutes), indicating a high likelihood that thedetected precursor ion is in fact SFLLALPAPLVTPEASAEAR* (SEQ ID NO:6).

Per the present method, the detection of product ions having an m/z ofthe y₁ ion generated by dissociation of the SFLLALPAPLVTPEASAEAR* (SEQID NO:6) precursor ion, triggered acquisition of a second,high-resolution MS/MS spectrum (in the orbital trapping mass analyzer)for measurement of product ions of the endogenous SFLLALPAPLVTPEASAEAR(SEQ ID NO:5) peptide. The ion chromatogram at lower right depicts ionintensities at the m/z's of the y₁ product ions of both the endogenousSFLLALPAPLVTPEASAEAR (SEQ ID NO:5) peptide and its isotopically-labeledSFLLALPAPLVTPEASAEAR* (SEQ ID NO:6) counterpart. It can be discernedthat peaks appear for both product ions, again centered around 54.8minutes retention time, thereby enabling quantitation of the amount ofthe SFLLALPAPLVTPEASAEAR (SEQ ID NO:5) peptide in the sample.

While the foregoing examples describe a mass spectrometry method for usein the analysis of peptides in a sample, those skilled in the art willrecognize that the method may be readily adapted to detect and measureother analytes, including substances of both biological andnon-biological origins.

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims.

What is claimed is:
 1. A method for analyzing a peptide-containingsample by mass spectrometry, comprising: (a) preparing the sample foranalysis, including adding a plurality of isotopically labeled peptidesto the sample, each one of the isotopically labeled peptides producing,when ionized, a labeled peptide precursor ion of characteristicmass-to-charge (m/z) ratio; (b) chromatographically separating thesample; (c) ionizing the separated sample to generate sample ions; (d)performing an MS1 scan of the sample ions; (e) identifying a precursorion in the MS1 scan that has a mass-to-charge ratio (m/z) matching anm/z of a labeled peptide precursor ion; (f) performing a first MS/MSanalysis of the identified precursor ion to acquire a first MS/MSspectrum; (g) determining whether at least one peak is present in thefirst MS/MS spectrum matching one or more m/z's of a characteristicproduct ion or product ions generated by dissociation of the labeledpeptide precursor ion, wherein the characteristic product ion or productions consists of no more than three product ions; and (h) upondetermination that the at least one peak is present in the first MS/MSspectrum, performing a second MS/MS analysis to measure an intensity ofat least one product ion produced by dissociation of the endogenouspeptide ion corresponding to the labeled precursor ion.
 2. The method ofclaim 1, wherein steps (d) and (h) are performed in a first massanalyzer, and step (f) is performed in a second mass analyzer separatefrom the first mass analyzer.
 3. The method of claim 2, wherein thefirst mass analyzer is an orbital electrostatic trap mass analyzer, andthe second mass analyzer is a quadrupole ion trap mass analyzer.
 4. Themethod of claim 1, wherein the characteristic product ion or productions is exactly one characteristic product ion.
 5. The method of claim4, wherein the one characteristic product ion is the y₁ product ion ofthe identified labeled peptide precursor ion.
 6. The method of claim 1,wherein step (g) comprises determining whether the at least one peak hasan intensity exceeding a threshold value.
 7. The method of claim 1,wherein step (g) comprising determining whether the product of theintensity of the at least one peak and the measured intensity of theprecursor ion measured in step (d) exceeds a threshold.
 8. The method ofclaim 1, wherein step (g) comprises determining whether a ratio of theintensity of the at least one peak to the measured intensity of theprecursor ion measured in step (d) is within a predetermined range ofvalues.
 9. The method of claim 1, wherein step (h) further comprisesmeasuring an intensity of at least one product ion produced bydissociation of the identified precursor ion in a third MS/MS analysis.10. The method of claim 9, wherein the product ion populations producedby the second and third MS/MS analyses are analyzed concurrently. 11.The method of claim 1, wherein step (f) is performed over a narrow m/zwindow in which the m/z of the at least one characteristic product ionis located.
 12. The method of claim 1, wherein step (e) comprisesdetermining whether an ion species present in a spectrum produced in theMS1 scan matches a value on a stored inclusion list.
 13. The method ofclaim 11, wherein the stored inclusion list is adjusted based onchromatographic retention time.
 14. The method of claim 2, wherein thesecond mass analyzer is a quadrupole mass filter.
 15. The method ofclaim 1, wherein step (d) is performed in a high resolution/accuratemass (HRAM) mass analyzer having a resolving power greater than 50,000and a mass accuracy better than 10 ppm.
 16. The method of claim 1,wherein step (e) comprises identifying whether two or more precursorions are present in the MS1 scan having m/z's that match m/z's of alabeled precursor ions at different charge states.
 17. The method ofclaim 11, wherein the narrow m/z window has a width of 2 m/z.
 18. Themethod of claim 1, wherein the peptide-containing sample is a biologicalfluid.